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Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics [1]

['Madeline G. Dans', 'Burnet Institute', 'Melbourne', 'Victoria', 'School Of Medicine', 'Institute For Mental', 'Physical Health', 'Clinical Translation', 'Deakin University', 'Waurn Ponds']

Date: 2023-04

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

Funding: This work was supported by the National Health and Medical Research Council (2001073 to P.R.G and W.N) and (119780521 to B.S.C), the Victoria Operational Infrastructure Support Programs received by the Burnet Institute and Walter and Eliza Hall Institute, the Academy of Finland (322917 to I.K and H.P), the Sigrid Jusélius Foundation (to I.K.) and the Hospital Research Foundation (to D.W.W). This work was also funded by an Australian Government Research Training Program Scholarship (to M.G.D), a University of Melbourne Research Scholarship (to T.K.J), an Ellen Corin Fellow (to B.E.S) and an National Health and Medical Research Council Senior Research Fellowship (1136300 to TdK-W). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Recently, we identified a compound MMV020291 (MMV291) from the Medicines for Malaria Pathogen Box as an inhibitor of P. falciparum merozoite invasion of RBCs [ 52 ]. We further explored the drug development potential of the compound by defining the structure activity relationship (SAR) and generated analogues with improved potency, while maintaining compound selectivity and invasion blocking activity [ 53 ]. Here, through in vitro resistance selection, whole-genome analysis, and reverse genetics, we show that the mechanism of resistance to MMV291 are through mutations in PfPFN and PfACT1. We further explore the MoA of the MMV291, which is the first reported compound series linked to interference with the actin-profilin complex in P. falciparum.

In apicomplexan parasites, actin polymerisation has been difficult to study because of the short length and instability of the actin filaments [ 14 , 41 , 42 ]. Despite this, actin polymerisation has been shown to be essential in many phases of the lifecycle, including intracellular replication, host cell egress (only in T. gondii), motility, and host cell invasion [ 14 , 17 , 43 , 44 ]. Of these, host cell invasion is the best-characterised actin-dependent process (reviewed in [ 44 ]). In P. falciparum, naturally occurring compounds such as cytochalasins D and B, latrunculins, phalloidin, and jasplakinolide have been used to study the complex actin regulation in merozoite invasion [ 7 , 29 , 45 – 48 ]. These compounds interfere with actin treadmilling by affecting the polymerisation and depolymerisation of actin through various MoA. For example, cytochalasins bind to the barbed end to prevent polymerisation and can also prevent G-actin disassociation from F-actin [ 49 ]; latrunculins prevent sequestration of the G-actin subunits [ 50 ]; and phalloidin and jasplakinolide stabilise F-actin by preventing the release of P i from G-actin-ADP subunits [ 51 ]. Apart from the recently developed latrunculins that have greater selectivity [ 47 ], these naturally occurring compounds remain biological tools rather than antimalarial candidates due to their cytotoxicity.

In comparison, actin is more conserved between apicomplexans and higher eukaryotes; however, the apicomplexan actins are among the most diverged actins in eukaryotes. P. falciparum encodes 2 actin isoforms: actin-1 (PfACT1) (PF3D7_1246200) and actin-2 (PfACT2) (PF3D7_1412500), the latter of which is only expressed in the sexual stages [ 40 ]. PfACT1 has a high degree of sequence identity (93%) with the single actin gene in T. gondii [ 20 ] and shares an 82% identical sequence with the human cytosolic β actin [ 14 , 29 , 40 ].

Profilin is essential for merozoite invasion of RBCs [ 36 , 37 ] and is required for efficient sporozoite motility [ 38 , 39 ]. Apicomplexan profilin has diverged markedly from higher eukaryotes [ 36 ], with low sequence identity (16%) retained between P. falciparum profilin (PfPFN) (PF3D7_0932200) and Homo sapiens profilin I (HsPFNI) sequences. This is illustrated by a unique arm-like β-hairpin insertion in apicomplexan profilin that spans from residues 57 to 74 [ 36 ]. This unique sequence is essential for actin binding; mutations in this region hinder monomer sequestration in vitro and impact sporozoite motility [ 38 ]. Within apicomplexan, PfPFN has also diverged from Toxoplasma gondii profilin (TgPFN), with the former acquiring a further acidic loop extension located in residues 40 to 50 [ 36 ].

The generation of filamentous actin (F-actin) is required for both gliding motility and RBC invasion by the parasite [ 22 , 23 ]. F-actin is formed through the incorporation of subunits of globular actin (G-actin)-ATP at the barbed end. To stabilise the growing filament, hydrolysis of G-actin-ATP occurs to generate G-actin-ADP and inorganic phosphate (P i ) [ 24 – 26 ]. For filament disassembly and G-actin turnover, the release of P i at the pointed end destabilises the F-actin, resulting in the disassociation of G-actin-ADP [ 27 , 28 ]. The continuous growth at the barbed end and shortening at the pointed end of the filament is referred to as actin treadmilling [ 25 ] and is a process that is tightly regulated by a plethora of actin-binding regulatory proteins, many of which are absent in apicomplexan parasites [ 14 , 29 ]. However, one key protein present across eukaryotes from Apicomplexa to Opisthokonts is profilin, a sequester of G-actin [ 30 ]. The role of profilin is to maintain a pool of polymerizable actin monomers and to catalyse the exchange of ADP to ATP in the monomers [ 31 , 32 ]. Profilin then delivers these G-actin-ATP to formin, a nucleator and processive capper that binds to the barbed end of the actin filament [ 33 – 35 ].

One unique process required for parasite invasion of RBCs is the engagement of an actomyosin motor complex, termed the glideosome, a mechanism that is shared between apicomplexan parasites. In the glideosome, a single-headed class XIV myosin A, MyoA, is tethered to the inner membrane complex of the merozoite via its 2 light chains and several glideosome-associated proteins [ 10 – 12 ]. MyoA produces the force required for gliding motility by walking along filamentous actin [ 13 – 16 ]. Actin filaments, in turn, are linked to surface exposed adhesin proteins via the glideosome-associated connector [ 17 ]. A ring of adhesions, termed the tight junction, is then formed between the apical tip within the merozoite and the RBC, and the force produced by the MyoA power stroke propels the merozoite into the RBC through the established tight junction [ 18 , 19 ]. This movement translocates the tight junction from the apical to the posterior end of the parasite and results in the RBC membrane enveloping the merozoite that later forms the PV [ 5 , 20 , 21 ].

The red blood cell (RBC) stage of Plasmodium infection within the human host leads to the exponential growth of the parasite and the symptoms of the disease. Within RBCs, parasites develop within a parasitophorous vacuole (PV) in a series of stages from rings to trophozoites and, finally, schizonts. During schizogony, daughter merozoites are formed, which eventually egress from the RBC to reinfect new RBCs. The invasion of RBCs by merozoites is a complex and finely tuned process that involves a multitude of unique signalling cascades and protein–protein interactions [ 5 ]. These events must occur within minutes of a merozoite egress to allow successful RBC internalisation, and, therefore, this process represents a prime opportunity against which to develop therapeutics [ 6 – 9 ]. Administering an invasion inhibitor in combination with a drug targeting intracellular parasite processes would span the entire asexual blood stage of infection and could effectively prevent parasite proliferation [ 8 ].

Malaria is a devastating parasitic disease that caused approximately 619,000 deaths in 2021, an upward trend from 2020’s figure of 558,000 due to COVID-19-related service disruption [ 1 ]. The majority of these deaths were a result of infection with Plasmodium falciparum, which causes widespread disease across sub-Saharan Africa. The alarming increase in deaths, combined with the spread of mutations conferring resistance to frontline antimalarials [ 2 – 4 ], highlights the urgent need to find new compounds with unique mechanisms of action (MoAs) to combat this deadly parasite.

While all these data pointed to the MMV291 series having specificity for the asexual stage of P. falciparum infection, mostly during merozoite invasion, an outstanding question important for drug development was whether this chemotype disrupted actin filaments in eukaryotic cells. To address this, we labelled F-actin in HeLa cells and exposed them to the classical actin inhibitors, Latrunculin B and CytD, and increasing concentrations of MMV291 before imaging them by lattice light shield microscopy across 3 hours ( S13 Fig ). This revealed that the actin inhibitors, Latrunculin B and CytD, had profound effects on disrupting actin filaments even after only 30 minutes of treatment ( S13B and S13C Fig and S1 Movie ). In comparison, MMV291 treatments (even up to the 20 μM concentration) were seen to have no effect on the filaments, with similar labelling seen to that of the vehicle control, DMSO ( S13A and S13D–S13G Fig and S1 Movie ). Altogether, this demonstrates that the MMV291 chemotype is specific for disrupting P. falciparum actin.

Defects in apicoplast inheritance for daughter merozoites induce a “delayed death phenotype” whereby drugs targeting the apicoplast, such as the antibiotic azithromycin, exhibit no parasiticidal activity until the second cycle of growth after defective merozoites invade new RBCs and progress to trophozoites [ 67 – 69 ]. To investigate if MMV291 also produced a delayed death phenotype, highly synchronous ring-stage parasites expressing an exported nanoluciferase protein were treated with a titration of azithromycin, chloroquine, or MMV291. After 40 hours and prior to merozoite invasion, the compounds were washed out and parasites allowed to grow for a further 2 cycles with nanoluciferase activity used as a marker for parasite growth ( Fig 6B ). This demonstrated that azithromycin-treated parasites in cycle 1 elicited a dose–response decrease in parasite biomass in cycle three, producing an EC 50 of 67.5 nM ( Fig 6Ci and 6Civ ), in contrast to chloroquine, which demonstrated the profile of a fast-acting antimalarial ( Fig 6Cii ). MMV291 displayed some intermediate delayed death activity at the maximum concentrations tested with cycles 2 and 3 producing EC 50 ’s of >10 μM and 7 μM, respectively ( Fig 6Ciii and 6Civ ). This was significantly higher than the compound’s overall EC 50 of 0.5 to 0.9 μM in a 72-hour LDH assay, suggesting apicoplast segregation and subsequently delayed death is a secondary MoA of MMV291.

(A) i Representative panels from live cell imaging of apicoplast targeted acyl carrier protein (ACP) tagged with GFP revealed that treatment of trophozoites for 24 hours with both 5 μM and 10 μM MMV291 (10 and 20 × EC 50 ) disrupted apicoplast segregation, resulting in an increase in abnormal apicoplast clumping at schizonts. Scale bar indicates 5 μm. ii These images were quantified by 3 independent blind scorers, which showed a significant decrease in the normal segregation of apicoplasts between the 10 μM MMV291 and DMSO treatments, while the 5 μM MMV291 was not significant (ns). The number of cells analysed were 60, 48, and 47 for DMSO, 5 μM MMV291, and 10 μM MMV291, respectively, which were captured over 3 biological replicates. The error bars represent the standard deviation of 3 independent blind scoring. Statistics were performed via a two-way ANOVA using GraphPad Prism between the DMSO segregated panel and the other treatments. *** indicates P < 0.001. (B) Schematic of the delayed death assay set-up. Around 0–4 hours postinvasion (hpi) ring-stage parasites expressing nanoluciferase (Nluc) were exposed to titrations of compounds for approximately 40 hours before compounds were washed out. Each cycle for 3 cycles, samples were collected for evaluation of Nluc activity to quantify parasitemia. ( C) Azithromycin (i) , chloroquine (ii) , and MMV291 (iii) were evaluated in the delayed death assay where it was found that unlike azithromycin, MMV291 did not display characteristics of a delayed death inhibitor but had partial reduction in parasite growth at the highest concentration used (10 μM) in the second and third cycles. In contrast, the fast-acting antimalarial chloroquine exhibited killing activity in the first cycle. Growth was normalised to that of parasites grown in 0.1% DMSO and EC 50 values (iv) were derived from dose–response curves plotted from nonlinear regressions in GraphPad Prism with 95% confidence intervals of these values specified in brackets. Source data can be found in S1 Data .

Next, we examined the effect of MMV291 on other F-actin-dependent processes in the asexual stage. Conditional knockout of actin-1 in P. falciparum results in a defect in apicoplast segregation [ 65 ]. To evaluate the activity of MMV291 against apicoplast segregation, a parasite line that expresses a fluorescently tagged protein destined for trafficking to the apicoplast (ACP-GFP) was utilised [ 66 ]. Trophozoites were treated with 5 μM and 10 μM MMV291 (equating to 10 × EC 50 and 20 × EC 50 ) or the vehicle control before being imaged at schizont stages ( Fig 6Ai ). The schizonts were scored to either have apicoplasts that were reticulated (an immature branched form), segregated (mature form), or clumped (abnormal) [ 65 , 66 ]. This revealed that the DMSO treatment resulted in a majority of normal apicoplast segregation with GFP labelling visualised as distinct punctate signals in daughter merozoites ( Fig 6Aii ). In contrast, both concentrations of MMV291 induced a defect in apicoplast segregation whereby the 10 μM MMV291 resulted in significantly less segregated apicoplasts than the vehicle control (P = 0.0003; Fig 6Aii ). This was visualised as distinct “clumps,” reminiscent of the phenotype shown previously in actin-1 knockouts ( Fig 6A ) [ 65 ]. While the 5 μM concentration also displayed less segregation, this number was not significant (P = 0.18; Fig 6Aii ). Altogether, this indicated that MMV291 induced a dose response effect on apicoplast segregation.

F-actin is required for many processes across the lifecycle of P. falciparum including sporozoite gliding motility and hepatocyte invasion [ 38 , 64 ]. However, when sporozoites were treated with MMV291, both of these processes remained unaffected ( S10 Fig ). Similarly, despite the conserved sequences of actin-1 and profilin in P. falciparum and T. gondii ( S11 Fig ), MMV291 and its analogues also had little activity against tachyzoite invasion, unless the compounds were used at high concentrations relative to those used against P. falciparum (500 to 1,000 μM) ( S12 Fig ). Altogether, this, combined with previous data showing MMV291 has little activity against gametocytes [ 53 ], indicates that this compound series has activity solely in the asexual stage of P. falciparum infection.

To address whether the MMV291 analogues affected the PfPFN–PfACT1 interaction, we included PfPFN in the sedimentation assays. As PfPFN sequesters G-actin, only 21% of PfACT1 remained in the polymerised pellet fraction following sedimentation (Figs 5B and S9B ). In the presence of the compounds, the amount of PfACT1 in the pellet decreased significantly to 7.5% to 15% with S-MMV291, R-MMV291, S-W936, R-W936, and S-W414 treatment (P < 0.01; Figs 5B and S9B ). S-W827 exhibited the greatest affect by decreasing the PfACT1 to approximately 5% (P < 0.0001). The magnitude of the effects observed from the different compounds on actin sedimentation was correlated with the EC 50 values of the MMV291 analogues ( S8 Fig ) with the most potent inhibitors of parasite growth causing the greatest reduction in PfACT1 polymerisation. Notably, R-MMV291 had the smallest affect in agreeance with the weak parasite activity of this isomer compared to S-MMV291. In summary, these results indicate that the compounds act through a PfPFN-mediated mechanism to interfere with actin polymerisation in parasites.

PfACT1 (4 μM) under polymerizing conditions was quantified in the supernatant and pellet fractions in the presence of the MMV291 analogues (25 μM) or DMSO and upon addition of PfPFN (16 μM). (A) In the absence of PfPFN, 80 ± 4% of PfACT1 sedimented to the pellet fraction with the vehicle DMSO treatment. S-W936 decreased the amount of actin in the pellet to 68 ± 7%, while the remaining compounds had no significant effects on actin sedimentation. (B) Upon addition of PfPFN, actin sedimentation decreased to 21 ± 1% with DMSO treatment. All MMV291 analogues, S-MMV291, R-MMV291, S-W936, R-W936, S-W414, and S-W827, decreased the amount of actin in the pellet further to 11 ± 1%, 15 ± 2%, 8 ± 2%, 10 ± 4%, 9 ± 4%, and 5 ± 2%, respectively. Results are plotted as mean ± standard deviation of the relative amounts of actin in the pellet fraction. The data are based on at least 3 independent assays each performed in triplicate. Statistical significances were determined using an unpaired two-tailed t test, where ** P ≤ 0.01 and *** P ≤ 0.001, and **** ≤ 0.0001. No bar indicates not significant. Source data can be found in S1 Data .

Resistance to MMV291 arose due to mutations in both PfACT1 and PfPFN, suggesting the MMV291 series was interacting at the binding interface of the 2 proteins. To dissect the basis of this interaction, in vitro sedimentation assays with recombinant monomeric PfACT1 were carried out in the presence of compounds S-MMV291, R-MMV291, S-W936, R-W936, S-W414, and S-W827 and vehicle control, DMSO. In agreement with previous studies [ 42 , 63 ], 80% of PfACT1 could be sedimented into a pellet fraction by ultracentrifugation in the absence of MMV291 analogues (Figs 5A and S9A ), while 15% of PfACT1 could be sedimented in the nonpolymerizing (G-buffer) conditions ( S9C and S9D Fig ). S-W936 was found to cause a small but significant reduction in the amount of PfACT1 in the pellet to 68% (P = 0.01; Fig 5A ). The remaining compounds had no statistically significant effect on PfACT1 sedimentation. These results indicate that the MMV291 analogues have either no or minimal impact on actin polymerisation in vitro.

(A) Synchronised schizonts from a P. falciparum parasite line expressing an F-actin-binding chromobody were incubated with DMSO, Cytochalasin D (CytD), MMV291, and analogues S-W936 and R-W936, for 20 minutes at 37°C to allow merozoite egress. Merozoites were then imaged to detect either a normal punctate apical F-actin fluorescence signal or uniform signal, indicative of the inhibition of F-actin formation. Arrow heads depict punctate F-actin signal, and scale bar indicates 2 μm. ( B) The proportion of merozoites with a punctate or uniform signal were scored with >550 merozoites counted for each treatment. Merozoites treated with the lower concentrations of the less active R-W936 had equal proportions of punctate and uniform fluorescence signals, like the DMSO control. In contrast, CytD, MMV291, and the active S-W936 compounds all greatly inhibited the formation of a punctate F-actin signal. Error bars represent the standard deviation of 2 biological replicates, each made up of 3 technical replicates from 3 individual counters. Statistical analysis was performed using a one-way ANOVA, comparing the mean of CytD punctate proportions with the mean of other treatments. *** indicates P < 0.001; no bar indicates not significant. DMSO and CytD were used at concentrations of 0.1% and 1 μM, respectively. Source data can be found in S1 Data .

F-actin detection in apicomplexan parasites has been technically challenging because of the short length of the filaments produced [ 48 , 61 ]. Recently, this has been overcome with the expression of F-actin binding chromobodies in T. gondii [ 43 ] that have also been adapted to P. falciparum [ 62 ]. The actin binding chromobodies consist of an F-actin nanobody fused to green fluorescent protein to allow microscopic detection of F-actin, which exists as a distinct punctate signal located at the apical tip of the merozoite. With actin polymerisation inhibitors, such as CytD, the punctate fluorescence dissipates into a uniform signal across the merozoite [ 62 ]. To ascertain if MMV291 could inhibit actin polymerisation in merozoites, we treated synchronised schizonts expressing the fluorescent nanobody with the parent MMV291 molecule and 2 analogues; S-W936, an active S-stereoisomer (EC 50 of 0.2 μM), and R-W936, a less active R-stereoisomer of the former molecule (EC 50 of 6.9 μM) ( S8 Fig ) at both 5× or 1× the growth EC 50 ( Fig 4 ). R-W936 was also tested at multiples of S-W936 EC 50 to allow a direct comparison between the compound’s activity and effect on actin polymerisation. Images of the egressed merozoites were captured and quantification of the punctate versus uniform F-actin signal was scored ( Fig 4A ). This revealed that at both concentrations of MMV291 and S-W936 tested, and high concentrations of less active isomer, R-W936, caused a similar reduction in merozoites expressing F-actin puncta to CytD treatment (P > 0.05; Fig 4B ). In contrast, low concentrations of the less active isomer, R-W936, was significantly less effective at preventing merozoites from forming F-actin puncta than CytD (P < 0.001; Fig 4B ). This result was notable as it provides the first direct link between the parasiticidal activity of MMV291 and its ability to inhibit F-actin formation in merozoites.

Two clones from 3 independently derived MMV291-resistant parasite lines were tested in 72-hour LDH growth assays. Varying degrees of resistance to S-W827 (A) , S-W936 (B) , S-W414 (C) , and S-W415 (D) was observed, with Population C clones demonstrating the greatest resistance and Population B clones retaining the most sensitivity to the 4 molecules. Values were normalised to parasite growth in 0.1% DMSO, with error bars representing the mean of 3 biological replicates. Dose response curves were generated in GraphPad Prism using nonlinear regression to derive mean EC 50 values, which are stated in the table. S.D indicates the standard deviation calculated from EC 50 values across 3 biological experiments. Heat map indicates degree of resistance from 3D7 control lines, with yellow and red indicating the lowest and highest degree of resistance, respectively. Source data can be found in S1 Data .

We have previously reported the SAR of MMV291 whereby the alpha-carbonyl S-methyl isomer was determined to be important for parasiticidal activity [ 53 ]. Four of these analogues, S-W414, S-W936, S-W415, and S-W827 ( S8 Fig ) (previously referred to as S-18, S-20, S-22, and S-38) were selected to study the relationship of the chemical series targeting PfACT1 and PfPFN. These S-stereoisomers of the racemic MMV291 compound were tested on 2 clones from each chemically induced MMV291-resistant population in a 72-hour LDH growth assay. This revealed that the resistant clones maintained their resistance against the potent analogues of MMV291 ( Fig 3A–D ). Interestingly, the degree of resistance differed depending on the parental population; population B clones (PFN(K124N)) were the least resistant, inducing a 10-fold increase in EC 50 in the 4 analogues, while the population C clones (ACT1(M356L)) exhibited the most resistance, increasing the EC 50 60 to 170-fold. These data indicated that since the ACT1(M356L) clones were consistently highly resistant to the MMV291 analogues, the MoA of this chemical series may be linked to PfACT1 function.

To date in P. falciparum, the dynamics of actin polymerisation have been explored with naturally occurring compounds that bind to various regions within the actin-1 protein [ 7 , 45 – 48 , 51 , 58 ] ( S4 Fig ). Targeting the actin-binder profilin, however, presents a novel mechanism to interfere with this essential parasite process. This novel MoA of MMV291 was confirmed by the lack of cross-resistance between the chemically induced MMV291-resistant parasites and cytochalasin D (CytD) and jasplakinolide in a 72-hour LDH growth assay ( S5 Fig ). Furthermore, despite the highly conserved sequence of actin-1 in H. sapiens and P. falciparum ( S6 Fig ) and that actin dynamics in RBCs have previously been shown to be perturbed by actin inhibitors [ 59 , 60 ], RBCs that had been pretreated with MMV291 displayed normal levels of merozoite invasion, indicating this compound is not targeting host actin ( S7 Fig ). Altogether, these data indicate that MMV291 has an alternative MoA from traditional actin polymerisation inhibitors.

(A) i Strategy to create the donor plasmid to introduce PFN(N154Y), PFN(K124N), and ACT1(M356L) SNPs into 3D7 parasites. Homology regions (HRs) were designed to the 5′ flank (HR1) and 3′ flank (HR2) whereby HR1 was made up of the endogenous genes’ sequence (HR1A) and recodonised fragments (HR1B), encompassing the resistant mutation alleles. A synthetic guide RNA (gRNA) was designed for either profilin or actin-1 to direct Cas9 to the cleavage site and induce double crossover homologous recombination. WR99210 was used to select for integrated parasites via the human hydrofolate reductase (hDHFR). ii Integration into the profilin or actin-1 locus was validated whereby a 5′ UTR primer (i/v) was used in combination with a primer located in the glmS region (k). B) i Integrated parasites were tested in a 72-hour LDH growth assay, which revealed the resistant mutations conferred resistance against MMV291 and confirmed the profilin and actin-1 proteins as involved in the MoA of the compound. Growth has been normalised to that of parasites grown in 0.1% DMSO, and error bars indicate the standard deviation of 3 biological replicates. Source data can be found in S1 Data . ii EC 50 values derived from nonlinear regression curves in GraphPad Prism with 95% confidence intervals shown in brackets. und = undefined.

To confirm that the chemically induced PfPFN(N154Y), PfPFN(K124N), and PfACT1(M356L) mutations were responsible for resistance to MMV291, we employed reverse genetics to introduce each mutation into wild-type (WT) parasites. This was achieved using a CRISPR-Cas9 gene editing system whereby homology regions were designed to both the 5′ and 3′ flanks of the desired loci. The 5′ homology flank encompassed a synthetic recodonised region containing the WT or nonsynonymous drug-resistant mutations and synonymous shield mutations to prevent recleavage with Cas9 after recombination into the desired loci ( Fig 2Ai ). To direct Cas9 cleavage, a homologous synthetic guide RNA (gRNA) was mixed with a tracrRNA and recombinant Cas9 enzyme and electroporated into blood stage parasites with the donor plasmid [ 57 ]. After chromosomal integration was selected with WR99210, viable parasites for both the mutant and WT parasites were confirmed to contain the donor cassette using integration PCRs ( Fig 2Aii ). These PCR products were sequenced and confirmed to contain the corresponding MMV291-resistant alleles ( S3 Fig ). Next, the modified lines were tested in an LDH growth assay against MMV291, which showed an 11- to 18-fold increase in EC 50 in the introduced mutant lines compared to their WT counterparts ( Fig 2B ). This indicated that PfPFN(K124N), PfPFN(N154Y), and PfACT1(M356L) were responsible for the chemically induced resistance by MMV291, suggesting these proteins are involved in the MoA of the compound.

A homology model of the binding of P. falciparum actin-1 (PfACT1) and profilin (PfPFN) was created using the binding of Orytolagus cuniculus actin to H. sapiens profilin [ 36 , 42 , 56 ] ( Fig 1D ). This indicated that PfACT1(M356) and PfPFN(N154) were located at the binding interface between the 2 proteins, while PfPFN(K124) was orientated away, on the opposite side of PfPFN. Despite the close proximity of these 2 SNPs to the binding site between the 2 proteins, the resistant parasites did not exhibit an associated fitness cost in vitro ( S2 Fig ), indicating these amino acid changes are well tolerated and may not be essential for actin-1 binding to profilin.

Across the 6 clones of MMV291-resistant parasites from 3 populations, there were a total of 18 nonsynonymous single nucleotide polymorphisms (SNPs) identified in 16 genes with no other gene variants found ( Table 1 ). Of these SNPs, 3 were present in related genes across all resistant isolates. One of these SNPs was located in chromosome (Chr) 12:1921849 within the gene encoding actin-1 (PF3D7_1246200), resulting in an M356L mutation in population C clones. The other 2 SNPs both occurred in the gene encoding profilin (PF3D7_0932200), located in Chr 9:1287853 and 1288316, resulting in a K124N and N154Y mutation in population B and D clones, respectively ( Fig 1C and Table 1 ).

(A) Chemical structure of MMV291. ( B) i Drug cycling on and off for 3 cycles and subsequent cloning out of parental lines resulted in 2 clones from 3 populations (Pop B, C, and D) that maintained stable resistance to MMV291 in a 72-hour growth assay. Growth has been normalised to that of parasites grown in 0.1% DMSO with error bars representing the standard deviation of 3 biological replicates. ii EC 50 values derived from nonlinear regression curves in GraphPad Prism with 95% confidence intervals shown in brackets. und = undefined. Source data can be found in S1 Data . ( C ) Genome sequencing of the MMV291-resistant parasites revealed a different nonsynonymous single nucleotide polymorphism (SNP) shared by the clonal lines across 2 related proteins; Populations D and B contained K124N and N154Y mutations in profilin (PF3D7_0932200), respectively, while Population C contained a M356L mutation in actin-1 (PF3D7_1246200). Scale bar indicates 100 base pairs. ( D ) The positions of the resistance mutations were mapped onto the X-ray structures of P. falciparum actin-1 (purple) (PDB: 6I4E) (42) and P. falciparum profilin (pink) (PDB: 2JKG) (36), which revealed PFN(N154Y) and ACT1(M356L) lie on either side of the proteins’ binding interfaces. PFN(K124N) resides on the opposing side of profilin. In this case, the X-ray structures of Oryctolagus cuniculus actin and human profilin (PDB: 2PBD) (56) were utilised as a template to spatially align the 2 parasite proteins.

To select for parasite resistance against our lead molecule MMV291 ( Fig 1A ), 5 populations of 10 8 P. falciparum 3D7 parasites were exposed to 10 μM (approximately 10 × EC 50 ) of the compound until new ring stage parasites were no longer observed by Giemsa-stained blood smears. The drug was removed, and parasites were allowed to recover. This drug on and off selection was performed for 3 cycles before parasite resistance was evaluated in a 72-hour lactate dehydrogenase (LDH) growth assay [ 54 ]. This revealed 3 MMV291-selected populations demonstrated an 8- to 14-fold increase in EC 50 ( S1 Fig ). These resistant populations (B, C, and D) were cloned out by limiting dilution, and 2 clones from each parent line were tested in an LDH assay, indicating resistance was heritable ( Fig 1B ). Genomic DNA was extracted from these resistant clones, along with a parental 3D7 reference strain, and whole genome sequencing was performed using the Oxford Nanopore MinION platform [ 55 ]. Here, a minimum of 10× depth coverage with 70% of the reads to support the called allele was required for verification [ 55 ].

Discussion

In this study, we sought to uncover the target and explore the MoA of a sulfonylpiperazine, MMV291, which acts to prevent merozoites from deforming and invading human RBCs. Resistance selection coupled with whole genome sequencing revealed 3 independent mutations in PfPFN and PfACT1 that did not impose a fitness cost on parasite growth in vitro. Furthermore, introducing these mutations into WT parasites mediated resistance to MMV291, indicating PfPFN and PfACT1 as proteins involved in the MoA of the compound. Interestingly, the 3 MMV291-resistant populations were observed to produce differing levels of resistance against the more potent MMV291 analogues, with parasites containing the PfACT1(M356L) mutation demonstrating the greatest resistance. This could indicate that MMV291 may interact with higher affinity to PfACT1 and thereby a conservative mutation may lead to reduced MMV291 binding, while still retaining the PfPFN–PfACT1 interaction. In contrast, the other 2 MMV291 PfPFN resistance mutations resulted in more radical amino acid changes and the fact that these mutants elicit similar overall parasite growth as the conservative PfACT1(M356L)-resistant parasites could indicate greater plasticity on the profilin side in PfPFN-PfACT1 binding.

We have previously reported that although MMV291-treated merozoites cannot deform and invade RBCs, the merozoites are still capable of irreversibly attaching to their target RBCs and can subsequently trigger echinocytosis [52]. These characteristics are similar to those reported with CytD, although this naturally occurring compound acts upon the actin filament itself to prevent polymerisation [7,49]. We previously noted that for RBCs with adherent MMV291-treated merozoites that the period of echinocytosis was greatly prolonged and that the adherent merozoites often produced pseudopodial extensions [52]. Both effects have recently been observed in CytD-treated merozoites utilising a lattice light shield microscopy system [70]. Here, membranous protrusions were described projecting from the parasite itself and extending into the interior of the RBC [70]. This CytD defect in merozoite invasion has also been reported as internal “whorls,” which were visualised with antibody staining of the rhoptry bulb protein RAP1, indicating that although CytD blocks merozoite entry, rhoptry release was unaffected [71]. Disruption of RBC integrity due to the injection of merozoite rhoptry contents therefore appears to cause extended RBC echinocytosis unless the merozoite can enter the RBC and reseal the entry pore.

To further investigate the MMV291 series effect on actin polymerisation, in vitro actin sedimentation assays were carried out, revealing the compounds had no activity against PfACT1 polymerisation in the absence of PfPFN, apart from S-W936 that caused a slight reduction. However, all compounds tested significantly enhanced the ability of PfPFN to sequester actin monomers, with the greatest effects observed for the analogues, which most potently inhibited parasite growth. It should be noted that although 2 of these analogues (R-MMV291 and R-W936) have low potency against the RBC stage of P. falciparum (EC 50 >11 μM and 6.9 μM, respectively), sedimentation assays were carried out at 25 μM, which could explain their activity in PfACT1 sequestration in the recombinant assay. This PfACT1 sequestration effect seen with the MMV291 analogues suggests that this compound series could stabilise the interaction between PfACT1 and PfPFN, leading to decreased actin polymerisation. This could have a profound impact on the formation and turnover of F-actin required for invasion and other cellular functions. Indeed, a downstream effect was observed in parasites expressing an F-actin chromobody whereby the MMV291 series was found to inhibit F-actin in merozoites in a manner that correlated with the parasiticidal activity of the compound. Altogether, this forms the basis of our proposed model of the MoA of MMV291, whereby MMV291 may increase the PfPFN sequestering effect of PfACT1, resulting in less PfACT1 turnover for the formation of the filaments, thereby functionally hindering the actomyosin motor and preventing merozoite invasion of RBCs (Fig 7). While the predictive model of bound PfPFN and PfACT1 places 2 of the 3 resistance mutations in the binding interface between the proteins, the exact binding location and subsequent “target” of MMV291 remains to be uncovered. Further studies into the compounds’ effects on the kinetics of actin polymerisation in parasites and crystallography studies solving the binding site of the MMV291 series in relation to the PfPFN-ACT1 interaction would be worthwhile attempting in order to confirm this stabilisation model and gain a greater understanding of the druggable potential of these essential parasite proteins.

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TIFF original image Download: Fig 7. Proposed model for MMV291 interference in profilin-mediated filamentous actin polymerisation. (A) Treadmilling model of profilin’s role in sequestering G-actin and stimulating the exchange of ADP for ATP before delivering the subunits to the barbed end of the growing filament. Here, formin initiates the polymerisation process to form F-actin. Hydrolysis of the G-actin-ATP occurs at this end to produce G-actin-ADP and inorganic phosphate (P i ), to stabilise the filament. The slow release of P i at the pointed end induces filament instability and proteins such as ADF1 bind to G-actin-ADP to aid in the release of the subunits, thereby severing the filaments. (B) A potential mechanism for MMV291’s inhibitory activity could be through the stabilisation of the G-actin/profilin dimer therefore inhibiting the formation of F-actin and preventing the generation of force required for invasion. ADF1, actin depolymerising factor 1; F-actin, filamentous actin; G-actin, globular actin. https://doi.org/10.1371/journal.pbio.3002066.g007

The proposed MoA of PfPFN-ACT1 stabilisation within parasites contrasts a previously identified inhibitor of the profilin1/actin interaction in mammalian cells that was found to combat pathological retinal neovascularization [72,73]. Here, the authors confirmed the competitive activity of the compound by demonstrating its inhibition of actin polymerisation in the presence of profilin1 [73], highlighting the druggable potential of this protein–protein interaction. It is thought that apicomplexan profilin may have originated from an evolution fusion of 2 ancestral genes [74], and, therefore, the PfPFN–ACT1 interaction may provide the basis of a selective drug target not found in their mammalian counterparts. This was reinforced by the lack of activity of MMV291 against HepG2 cells [53] or merozoite invasion into RBCs pretreated with MMV291. Additionally, we further extrapolated the selectivity of MMV291 for Plasmodium by confirming that the compound did not affect actin filaments in HeLa cells. Despite the phenotype of MMV291-treated merozoites phenocopying CytD, the MoA of MMV291 interference in actin polymerisation is more reminiscent of the latrunculins. These naturally occurring compounds prevent actin turnover through binding to G-actin subunits [47,50]. While targeting both G-actin and PFN-ACT1 result in a similar phenotype of reduced filament formation, compounds directed at the PfPFN–ACT1 interaction may have more success due to greater selectivity, a phenomenon we observed in our imaging of actin filaments in HeLa cells.

While crucial for merozoite invasion, PfPFN-PfACT1 may not be required for other F-actin-dependent processes such as gametocytogenesis and apicoplast segregation [14,65,75]. The MMV291 series have previously shown little activity against gametocytes [53,76], and while we did observe some activity against apicoplast segregation with MMV291 treatment, this parasiticidal activity occurred in much greater concentrations than observed within a standard 72-hour growth assay. This implicates apicoplast segregation as a secondary MoA of MMV291 and perhaps other G-actin sequestering-binding monomers, such as actin-depolymerisation factor 1 (ADF1) [77,78], could be the predominant PfACT1 sequesters that are utilised by parasites for these F-actin-dependent processes. Additionally, the requirements for PfACT1 sequestering and subsequent turnover of F-actin may vary dependent on the process at hand. This can be realised by the varying speeds in motility in different stages of parasites; ookinetes move at 5 μm/min [79–81], merozoites are the next fastest at 36 μm/min [23], while the fastest are sporozoites, which can reach speeds of 60 to 120 μm/min [82,83].

It was somewhat unexpected that MMV291 did not reduce sporozoite motility since sporozoites have been shown to be highly sensitive to mutations in profilin [38,39]. However, these mutations were located in an acidic loop and a conserved β-hairpin domain, which led to the disruption or weakening of the PFN-ACT1 complex and thereby implicating this interaction as an essential requirement for fast-gliding motility [38]. It is therefore possible that our proposed MMV291 MoA of stabilisation of the PFN-ACT1 interaction is not detrimental to actin polymerisation within sporozoites. Furthermore, we showed that hepatocyte invasion of sporozoites were unaffected by MMV291 treatment. This may be due to the different requirements for the PFN–ACT1 interaction to aid in actin polymerisation and subsequent G-actin turnover to invade these host cells with varying membrane tensions and elasticity. Altogether, this indicates this particular interference in the PfACT1–PfPFN interaction appears to specifically inhibit P. falciparum invasion of RBCs.

This trend of specificity for merozoite invasion of RBCs was extended to T. gondii where tachyzoites also displayed limited sensitivity to the MMV291 series. Here, high concentrations of compounds were required to elicit a reduction host cell invasion. Despite TgPFN being essential for host cell invasion [84] and that TgPFN and PfPFN proteins are somewhat conserved [38], a key difference between these parasites is that TgPFN inhibits the conversion of ADP-ATP on G-actin, thereby inhibiting F-actin polymerisation [85,86]. This has not been observed with PfPFN [14,38]. This highlights the diverged nature of profilin within apicomplexan parasites and, along with differences in host cells, may explain the disparity in activity of the series between P. falciparum and T. gondii.

Within the Plasmodium spp., profilin is highly conserved [36]. MMV291 has previously been shown to possess activity against Plasmodium knowlesi, albeit with less potency than P. falciparum [53], indicating that there may be a conserved PFN-ACT1 mechanism across Plasmodium spp. that is required for invasion of RBCs. Indeed, the resistant mutation locations are conserved in P. knowlesi profilin (PkPFN(K125), PkPFN(N155)) but further work as to whether this parasiticidal activity is linked to invasion defects in P. knowlesi, and if it extends to other Plasmodium spp., is required.

In summary, this investigation identified the first specific inhibitor of P. falciparum actin polymerisation in which its MoA could be linked through interference with PfPFN/ACT1 dynamics. The antimalarial development of this compound is currently hampered by the high clearance of MMV291 and its analogues in liver microsomes [53]. Additional medicinal chemistry work is therefore required to address the metabolic instability of this series before it can progress further towards a future antimalarial. Nonetheless, the MMV291 series could serve as a useful tool to study the complex regulation of actin polymerisation in the malaria parasite. This, in turn, could provide a starting point for future development of novel scaffolds against profilin-mediated F-actin polymerisation.

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